Accumulation and clearance of tissue residues and health status of Nile tilapia Oreochromis niloticus (L.) juveniles as influenced by the extended oral oxytetracycline-dosing

Antibiotics are considered an important primary therapy for bacterial diseases in aquaculture. This study evaluated the influence of oral administration of oxytetracycline (OTC) on feed intake, growth, mortality, residue accumulation and clearance, and histopathological changes in the vital organs of six groups of Nile tilapia Oreochromis niloticus when fed at 0–10 times the therapeutic dose (1×: 80 mg/kg biomass/day) for 10 and 20 consecutive days. The feed intake was reduced only slightly, viz., 2% in 10-day and 4.25% in 20-day dosing trials at 1× dose compared to control. While in other groups, an OTC-dose-dependent reduction in feed intake up to 31.25% was noted. The fish of the 0.5× and 1× groups recorded significantly high biomass, while the other OTC-dosed groups recorded significantly lower biomass than the control. The fold change in biomass between the control and 1× groups was insignificant. Dose-dependent mortalities were recorded in OTC-dosed fish in 10-day (1.67–6.67%) and 20-day (3.33–8.33%) trials. The OTC concentration in fish muscle established a dose- and time-response relationship. The OTC residue levels in muscle even on day 20 OTC-dosing were lower than the maximum residue limit (MRL) permitted by Codex Alimentarius (200 ng/g). On day 23 post OTC-dosing, the residue levels were traces to <10 μg/g in all groups, except the 10× group. The OTC-dosing caused mild to moderate pathological changes in the gills, liver and kidney of O. niloticus and the fish were able to mount adaptive biological responses to overcome the stress with time.


Introduction
The world food fish aquaculture production increased from 32.4 million tonnes in 2000 to 82.1 million tonnes in 2018 (FAO 2020). Tilapia (Oreochromis spp.) are the third most farmed and intensively cultured fish worldwide. Among the tilapia, production of Nile tilapia Oreochromis niloticus increased to 4.5254 million metric tonnes, with a share of 8.3% of the total world aquaculture production in 2018 (FAO 2020). The disease is one of the major predicaments that have an outcome on the tilapia production and livelihood of the farmers. Antibiotics are considered an important primary therapy for bacterial diseases in aquaculture (Hernandez 2005;Romero et al. 2012). Tetracyclines are widely used in the aquaculture industry because of their wide spectrum of antibacterial activity (Wen et al. 2006;Romero et al. 2012). The United States Food and Drug Administration (USFDA)approved antibiotic oxytetracycline (OTC) is the most widely used chemotherapeutics to treat specific and systemic bacterial infections of finfish (USFWS 2015;Julinta et al. 2019aJulinta et al. , 2019b. Many reports are available on the application of OTC and its usefulness in controlling bacterial diseases in aquaculture (Hernández 2005;Watts et al. 2017;Limbu et al. 2020). However, the abuse of antibiotics in animal husbandry and aquaculture, as well as lack of awareness on the withdrawal time after treatment, has resulted in their release into the surrounding environments and emergence of antibiotic resistance in pathogenic bacteria (Watts et al. 2017;Limbu et al. 2020). Furthermore, the presence of antibiotic residues in food fish might cause adverse reactions in some susceptible consumers (Romero et al. 2012;Watts et al. 2017;Limbu et al. 2020).
Across the world, the apprehension on the consumption of food fish treated with antibiotics has been increasing due to the impact on the economy, public health and consumers' ethics ). However, the information on direct human health risk through consumption of antibiotics contaminated food fish is lacking (Limbu et al. 2018(Limbu et al. , 2020. To ensure the safety of food supply for consumers and simplify international trade, the maximum residue limits (MRLs) for OTC residues in fish muscle have been established as 100 ng/ g by European Union [EU] (Anonymous 2010a) and 200 ng/g by the Codex Alimentarius (Anonymous 2006), Canada (Anonymous 2010b) and Japan (Anonymous 2011). Furthermore, the safety data on the farmed fish are required as part of the drug registration process in most countries. In fish, the drug metabolism is generally temperature-dependent, so is also the toxicity, which is greater at higher temperatures (Li et al. 2012). Also, the potential effects of medicated feed administration in tropical fish are limited. The aquaculture use of antibiotics is reportedly causing numerous effects on the developmental, cardiovascular and metabolic systems of fish than their primary purpose, which may vary with species and life stage, antibiotics types, mode of action, dose and dosages (Yang et al. 2020). Besides, the antibiotics may affect the growth performance of the cultured fish through enhanced oxidative stress, body damage, impaired nutrients metabolism, feed utilisation efficiency and immunosuppression (Limbu et al. 2020). In global aquaculture, at least eight different groups of antibiotics, dominated by tetracyclines, are used. Among them, OTC is the most abused (Limbu et al. 2020). This study, therefore, evaluated the effects of oral administration of OTC at different concentrations on the feed intake, growth, mortality, residue accumulation and clearance and pathological damages in the tissues of vital organs of O. niloticus juveniles when fed for 10 and 20 consecutive days at 22-26°C (22.82±0.78°C).

Materials and methods
Experimental fish, design and set up for 10 and 20 days of oxytetracycline-dosing trials The stocks of Oreochromis niloticus juveniles of size 10.39 ±0.67 cm and 13.40±0.48 g were collected locally from a grow-out system, packed in oxygen-filled bags and transported to the laboratory. The fish were acclimatised for 15 days in circular fibreglass reinforced plastic (FRP) tanks of 500-L capacity. Polypropylene tanks of size L58 × H45 × B45 cm were used for the feeding trials. The tanks were filled with 80-L bore-well water and conditioned for three days. Each tank was then stocked with 20 fish from the acclimatised stocks and protected by a nylon net cover. The rearing tank water quality parameters such as temperature (22.00-26.00°C), pH (7.80-8.60), dissolved oxygen (4.20-4.90 mg/ L), ammonia (0.003-0.008 mg/L), nitrite (0.13-0.73 mg/L) and nitrate (0.12-0.75 mg/L) were maintained optimally throughout the experimental period.
The safety of OTC in O. niloticus was evaluated through oral administration for 10 days (10-day trial) and 20 days (20day trial) separately. The therapeutic dose of OTC, as approved by the USFDA, is 2.50-3.75 g/100 pounds body weight/day (or 55-82.5 mg/kg biomass/day) for 10 consecutive days (USFWS 2015). The therapeutic dose of 80 mg/kg biomass/day was considered a 1× dose in this study. For each trial, the fish were grouped into 6 groups namely control (0×), 0.5×, 1×, 3×, 5× and 10× groups, in triplicate. The fish were fed with 2% of the body weight (BW) thrice daily. The faecal materials and other wastes were removed daily. About 50% of the water was exchanged once in 3 days.

Oxytetracycline feed preparation
Five medicated feeds with varied OTC concentrations were prepared by adding required quantities of oxytetracycline dihydrate powder (HiMedia, India) as described in our earlier studies (Julinta et al. 2019a(Julinta et al. , 2019b. In brief, the weighed OTC powder was mixed separately in 5-mL refined vegetable oil and then admixed with 1 kg basal pellet feed. The control feed was prepared as above without antibiotic. After proper mixing, the control and OTC feeds were air-dried under the fan for a day and stored separately in airtight plastic containers at room temperature. The experimental protocols fulfilled the ethical guidelines including adherence to the legal requirements of India (CPCSEA 2018).

Safety of oxytetracycline (OTC)-dosing
The total experimental period was 40 days. The fish of all the treatment groups were given the appropriate OTC feeds at 2% BW thrice daily to get a nominal therapeutic OTC dose of 40 mg (0.5×), 80 mg (1×), 240 mg (3×), 400 mg (5×) and 800 mg (10×)/kg biomass/day. The control group received the control feed (0×). The fish of 10-and 20-day trials were offered the control feed during the pre-dosing period (days 1-7). During the 10-day (days 8-17) and 20-day (days 18-27) dosing periods, the fish of 0.5×-10× groups were fed with appropriate OTC feeds. After the completion of dosing, i.e. the post-OTC-dosing period (days 18-40 for 10-day trial) and (days 28-40 for 20-day trial), control feed was offered to all the groups. The daily observations include mortality, external signs of infections and behavioural changes. The observed behavioural changes were the position of fish in the water column, gasping for air, flashing, hyperactivity, lethargy, loss of equilibrium, pigmentation or discolouration and other unusual behaviour or signs. The biomass of at least 10 fish from each group was measured on days 0, 10, 20, 30 and 40. The tank-wise uneaten feed pellets, if any, were collected after 90 min of each feeding, air-dried and weighed daily. Based on the relative amount of feed consumed daily, the feeding behaviour scores were established on a 5-point ordinal scale as per the descriptions of Bowker et al. (2015), i.e. 0: no feed consumed; 1: approximately 25% feed consumed; 2: approximately 50% feed consumed; 3: approximately 75% feed consumed and 4: approximately 100% feed consumed.
Oxytetracycline residue analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Six fish for each group, i.e. two fish from each of the replicate tank, were collected on day 0, day 10 and/or day 20 OTCdosing and on the last day of the experiment (day 40) for OTC residue analysis. The fish were put to death humanely using clove oil (50 μL/L), decapitated, dissected carefully, observed for gross lesions and clinical signs in the vital organs, degutted, washed thoroughly and stored at −20°C. The stored fish samples were packed in dry ice at a 1:3 ratio and transported to the Central Institute of Fisheries Technology, Kochi, India, for LC-MS/MS analysis. Immediately on reaching, the samples were kept in −20°C. Before the extraction of OTC, the fish samples were thawed and collected the edible muscle tissues with the skin. The OTC residues from 2 g of fish homogenates were extracted, purified and analysed as described in Sharma et al. (2020). The AB Sciex 4000 QTRAP mass spectrometer coupled with the Exion HPLC system was used for the residue analysis. The method of validation was as per the 2002/657/EC decision (European Commission 2002).

Histopathology
The kidney, liver and gill tissues of two fish from each of the tanks of 0× and 1× groups, collected for the LC-MS/MS analysis, were cautiously dissected out on day 0 and days 10 and/ or 20 OTC-dosing and processed for histopathology. This involved fixing the samples separately in Bouin's fixative, dehydrating the tissues to remove fixative using a series of alcohol gradation, dehydrating the tissues to remove alcohol, embedding in paraffin, sectioning at 5 μm using a rotary microtome and permanent slide preparation. The dried slides were stained by the haematoxylin and eosin (H&E) double staining method as described by Roberts (2012). Slides were permanently mounted using DPX (dibutyl phthalate xylene) mountant. The microphotographs were taken from the selected slides using an advanced Trinocular Research Microscope (Olympus, Japan, Model: BX51) fitted with a 16 MP SCO-LUX camera.

Statistical analyses
The data generated through triplicate experiments are presented as mean ± standard deviation. The non-parametric Kruskal-Wallis test with pair-wise comparisons was followed to analyse the feeding behaviour scores. The significance of differences in mortalities, biomass and OTC residue levels were examined by one-way ANOVA and Tukey HSD post hoc for the comparison of means followed by an independent sample t-test to assess the differences between days. Statistical Package for Social Sciences (IBM-SPSS) Version: 22.0 was used for the statistical analyses, considering a probability level of P<0.05.

Results
Effect of 10-day oxytetracycline-dosing on feed intake, mortalities and biomass The control and 0.5× groups were feeding aggressively, and there was no change in the feeding behaviour scores. The feed intake reduced with increasing OTC doses and the mean feeding behaviour scores decreased from 3.92±0.03 in 1× to 2.75 ±0.13 in 10×. The feed intake reduced significantly among the treatment groups in a dose-dependent manner as well as during the dosing period (P<0.05). During the post-dosing period, a significant improvement in feed uptake was noted (P<0.05), with scores in the range of 3.53±0.09 in 10× to 4.00±0.00 in the 1× groups (Table 1). Mortalities were noted only in the higher OTC-dosed groups (5× and 10×) during the periods of OTC-dosing. On day 10 OTC-dosing, the observed mortalities were 3.33 ± 2.89% and 6.67±2.89% in 5× and 10× groups. Within 3 days of cessation of OTC-dosing in 3× and 5× groups, an insignificant increase in mortalities was also noted. At the end of the experimentation, mortalities in the range of 1.67-6.67% were noted in 3×-10× groups ( Table 2). During the dosing period, the difference in mortalities of O. niloticus between 0× and 10× groups was significant (P<0.05). In the post-dosing period, the differences in mortalities among 0×, 5× and 10× groups were significant (P<0.05). The biomass of OTC-dosed O. niloticus juveniles showed an increment pattern from day 0. The fold increase in biomass differed significantly among the treatment groups (P<0.05). The fold increase in the biomass of fish fed the 0.5× and 1× doses were significantly high compared to 0× (2.32), with the highest at 0.5×, i.e. 2.51 times the initial weight. On the other hand, the fold increase in the biomass of fish fed the 3×-10× doses were significantly lower than the 0× (P<0.05). The 5× and 10×-dosed fish recorded the least growth rate, i.e. 2.11 and 2.03 times the initial weight, respectively (Table 3).
Effect of 20-day oxytetracycline-dosing on feed intake, mortalities and biomass Similar to the 10-day trial, reduced feed intake was noted in all cases compared to the pre-dosing period and/or control/0.5× groups. However, the reduction was more with the increase in the OTC-dosing period to 20 days. The mean feeding behaviour scores ranged from 2.60±0.10 (10×) to 3.83±0.06 (1×). Significant differences existed in the feed intake among the treatment groups and dosing periods (P<0.05). With the termination of OTC-dosing, the feed intake improved significantly in all the groups (P<0.05) and the scores ranged from 3.31±0.09 (10×) to 4.00±0.00 (1×; Table 1). No mortalities were observed in control and 0.5× groups until 40 days of observation. In the 1× group, only one fish died during the 20 days of OTC-dosing. The mortalities increased in a dosedependent manner with the highest in the 10× group (8.33 ±2.89%) on day 20 OTC-dosing. During the post-OTCdosing period, a slight increase in mortalities was noted only in 1× (3.33±2.89%) and 3× (5.00±0.00%) groups (Table 2). Significant differences in the mortalities of O. niloticus between the control and high OTC-dosed groups during the dosing (5×-10×) and post-dosing (3×-10×) periods were noted (P<0.05). Also, the differences in the mortalities between the 10-and 20-day dosing trials of all OTC-dosed groups (1×-10×) were insignificant (P>0.05). The fish of the 0.5× group recorded a significantly high fold change in biomass (2.45 times the initial weight) than the control (P<0.05). The fold Table 1 Feeding behaviour scores # of oxytetracycline (OTC)-dosed Oreochromis niloticus at 0-10 times the therapeutic dose of 80 mg/kg biomass/day (×) for 10 and 20 consecutive days   (Table 3). The biomass of the 0.5× group, though low in the 20-day trial, the difference was insignificant (P>0.05). The differences in the fold change in respective biomass of 1×-10× groups of 10-and 20-day trials were significant (P<0.05).

Abnormalities in oxytetracycline-dosed fish
The abnormalities on the skin, gills and intestine of OTCdosed O. niloticus were observed with the increase in OTC dose and dosing period. The skin of OTC-dosed fish was normal except for the 5× and 10× groups on day 20, and 10× on day 30, which had black patches on the skin. The gills of OTC-dosed fish appeared normal except for the 10× group on day 10-and 20-dosing, which exhibited reddening and excess mucus secretion. The intestines of the 5× group were discoloured on days 10 and 20, while those of the 10× group were discoloured as well as swelled on days 10 to 20. The swelling subsided on day 20 in 10-day trial and on day 30 in 20-day trial, but the discolouration of the intestine persisted. On and after day 30, all fish became normal and no abnormalities were observed. During the dosing period, the 3×-, 5×and 10×-dosed fish were gasping for air. All fish were distributed throughout the water column and there was no flashing, hyperactivity, lethargy and loss of equilibrium. The freshly dead fish were also subjected to necropsy, which had pale kidney and liver, discolouration and liquefaction of internal organs at the higher OTC doses.

Oxytetracycline residue accumulation and clearance
The residues in OTC-dosed O. niloticus juveniles for 10 and 20 days were analysed by LC-MS/MS. No OTC residues were found in control O. niloticus tissues as well as during the pre-dosing period. The residue levels increased in a dose-dependent manner from 63.71±3.82 μg/g in 0.5× to 397.15±12.33 μg/g in 10× groups on day 10 OTC-dosing. In 20 days of OTC-dosing, the levels of tissue residues increased from 94.52±4.12 μg/ g in 0.5× to 754.26±18.44 μg/g in 10× groups (Table 3). There were significant differences in OTC residue levels among the dosing groups and period (P<0.05). On the last day of the experiment, the recorded residue levels were traces to <10 μg/g in all groups, except for the 10× group. The residue levels were 12.78 ±1.61 μg/g and 85.20±3.69 μg/g for 10-and 20-day trials, respectively, on days 23 and 13 post OTCdosing (Table 3).

Discussion
The intensification of aquaculture favoured the use of a wide range of chemicals including several classes of antibiotics (Hernández 2005;Romero et al. 2012;Watts et al. 2017). The OTC is the most common one as it is an approved drug for aquaculture and has high potency against bacterial diseases (USFWS 2015). Drugs are assessed for the definition of their MRLs, and their environmental impact, safety and efficacy, which are relatively sparse in tropical aquatic species (Reimschuessel et al. 2005). Our results on O. niloticus indicated that the feed intake was reduced only slightly, viz., 2% in 10-day trial and 4.25% in 20-day trial at the therapeutic dose (80 mg/kg biomass/day) compared to control during the OTC-dosing period. On the other hand, a dosedependent reduction in feed intake with the increase in OTC concentrations from 10% in 3× to 31.25% in the 10× groups was noted. The reduced feed intake could be attributed to the action of OTC as a feeding deterrent, which can make feed less palatable (Toften and Jobling 1997;Limbu et al. 2020) and antibiotic-mediated toxicity (Zhang et al. 2015). An enhanced feed intake was noted in 3×-10× doses with the termination of OTC-dosing, yet it was 2.00-11.75% lower than the normal score of 4.0 in 10-day trial. A similar trend was also noted in 20-day trial, with a reduction of 17.00% in 3× to 35.00% in the 10× groups. Though the feed intake increased upon the termination of OTC-dosing, it was lower by 4.25% in 3× to 17.25% in the 10× groups than the control. Likewise, a significant dose-dependent reduction in feed intake was noted in O. niloticus when fed OTC (Julinta et al. 2019a;Limbu et al. 2020) or florfenicol (Gaikowski et al. 2013) for extended periods. On the other hand, Gaikowski et al. (2003) noted a slight reduction in feed consumption in Walleye (Sander vitreus) at the OTC dose of 413 mg/kg body weight/day, equivalent to the 5× dose of our study. Conversely, no doserelated effect on general fish behaviour or feeding behaviour was observed in juvenile sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis) when fed florfenicol-medicated feeds at 0, 15, 45 and 75 mg/kg BW/day for 20 days (Straus et al. 2012). Our results indicated that the feed intake may reduce by >2-fold if the dosing period is increased from 10 to 20 days or 5-fold if the therapeutic dose is increased by 3 times in healthy O. niloticus. It is a cause for concern when treating diseased fish. Mortalities were observed only from the 3× to 10× groups in 10-day OTC-dosing trial, with a maximum of 6.67±2.89% in the 10× group. On the other hand, dose-dependent high mortalities from 3.33±2.89% in 1× to 8.33±2.89% in 10× were recorded in 20-day OTC-dosed fish. Our results agree with Trushenski et al. (2018), who recorded 3% mortality in O. niloticus fed with 80 mg OTC/kg biomass/day for 10 days. Julinta et al. (2019a) also recorded increased mortalities with the increase in OTC dose, i.e. from 5.00±0.00% in 1× to 13.33 ±2.89% in 10× groups, respectively, when fed for 30 consecutive days. The observations like pale kidney and liver, discolouration and liquefaction of internal organs at the higher doses (3×-10× groups) possibly indicated the consequence of toxicity upon long-term oral OTC-dosing that might have triggered the mortality. It has been opined that any antibiotic/drug dose higher than the permissible limit may result in a toxicity reaction (Yang et al. 2020) or affect the parasympathetic nervous system (Limbu et al. 2020). The water quality parameters were optimally well within the acceptable ranges required for the normal growth of fish (Boyd 1979), which rule out stressful factors for the observed mortalities.
In both 10-and 20-day trials, the 0.5×-dosed O. niloticus recorded the highest growth rate, 2.45-2.51 times the initial weight, followed by 2.31-2.45 times in the 1× group. The lowest growth rate was recorded in 10× (1.99-2.03 times) Fig. 3 The histopathological sections of the a normal gill of O. niloticus juveniles, ×100 H&E staining; b 1×-dosed fish showing lamellar fusion (LF), curling of secondary lamellae (CSL) and desquamation (DS) on day 10 OTC-dosing, ×200 H&E staining; and c lamellar congestion (C), lamellar fusion (LF), desquamation (DS), lamellar epithelial lifting (EL), ruptured secondary lamellae (RSL), swelled lamellar tip (SLT) and thinning of secondary lamellae (TSL) on day 20 OTC-dosing, ×200 H&E staining followed by 5× (2.03-2.11 times) groups. The significant dose-dependent differences in the fold increase in biomass of O. niloticus among the dosing groups indicated that OTC at the higher doses may affect the growth so also in an earlier study with similar doses for 30-day dosing (Julinta et al. 2019a). The 0.5× dose had a significantly high growth increment, while the therapeutic dose (1×) did not affect the growth of O. niloticus. It has been proved in many earlier studies (Sanchez-Martinez et al. 2008;Reda et al. 2013) that OTC at lower doses favoured the growth of fish including tilapia, possibly due to the reduction of the gastrointestinal tract bacteria and their consequences (Dibner and Richards 2005). Contrarily, many earlier studies have shown the negative effects of antibiotics on feed digestibility (Toften and Jobling 1997), feed palatability and growth (Trushenski et al. 2018;Julinta et al. 2019aJulinta et al. , 2019bLimbu et al. 2020), immune system (Guardiola et al. 2012), serum biomarkers (Julinta et al. 2019b) and various other functions of fish (Limbu et al. 2020;Yang et al. 2020). The observed abnormal changes in OTCdosed O. niloticus at the higher doses also elucidated the side effects of long-term use of antibiotics, similar to several earlier studies that suggested OTC can induce nephrotoxicity and hepatotoxicity (Nunes et al. 2015;Julinta et al. 2019b;Limbu et al. 2020;Yang et al. 2020).
Residual periods of different antibiotics in various fish species are not well studied and, thus, have scarce information. Oxytetracycline remains one of the most common antibiotic residues found in animal tissues (Granados-Chinchilla and Rodríguez 2017). It is an easily degradable drug but can stay in muscle as metabolites or the pond sediment for a certain period (Romero et al. 2012;Watts et al. 2017). The absorption and elimination period is greatly influenced by temperature (Li et al. 2012). The present study at 22-26°C recorded a dose-dependent increase in OTC residues in edible muscles during the dosing period in both trials. Nevertheless, the residue levels were well within the MRL prescribed by Codex Alimentarius (200 ng/g; Anonymous 2006) or slightly above the EU regulations (100 ng/g; Anonymous 2010a) on day 10 OTC-dosing (119.75±6.72 ng/g) at the therapeutic dose (1×). The residue levels were traces to <10 μg/g in all groups, except the 10× group, i.e. 12.78±1.61 μg/g and 85.20±3.69 μg/g for 10-and 20-day trials, respectively, on the last day of the experiment. Likewise, many earlier studies noted residues of OTC in fish for several days or weeks (Reda et al. 2013;Elia et al. 2014;Limbu et al. 2020), which may pose an increased risk of the possible transfer of drug residues to the consumers (Mo et al. 2017;Limbu et al. 2020). Our results uphold the observations of Elia et al. (2014), who recorded significantly lower OTC residue levels in carp muscle 10 days after the antibiotic withdrawal than the MRL permitted by the EU regulation. Our residue clearance data may be useful to policymakers in establishing the best possible dosages or the withdrawal periods relevant to O. niloticus.
The renal toxicity may affect the tubules or glomeruli directly or alter hemodynamics indirectly, inflammatory tissue injury and/or obstruction of renal excretion (Seely et al. 2018). The disintegrated renal tubule, degenerated renal tubular epithelium and vacuolation were the major histological alterations observed in the kidney of 10-day OTC-dosed fish. Besides these, the extended dosing caused more alterations like disintegrated glomerulus, inflammation and necrosis of renal tubule, widened lumen and constricted renal tubule. The onset of necrosis and other inflammatory changes in the renal tubules may be the outcome of the toxic effect of longterm oral OTC-dosing and oxidative stress (Limbu et al. 2020). Nevertheless, the changes were observed to be only mild to moderate. The tubular epithelium degeneration noticed in the kidney indicated that the damage can be toxic, ischemic, inflammatory or obstructive. It has been reported that tetracycline interacts with organic anionic transporters (OATs) and mediates the renal excretion in the kidney, although tetracycline does not possess anionic moieties (Konig et al. 2013). These transport characteristics may lead to the accumulation of tetracycline and induction of tetracycline-associated nephrotoxicity (Yang et al. 2020). The lumen constriction in the collecting canals indicated a low filtration rate. The observations on the vacuolation in the renal tubules can reflect an alteration of many cytoplasmic components (Seely et al. 2018). Similarly, cytoplasmic vacuolisation in the kidney renal duct epithelium was demonstrated by Svobodova et al. (2006) in Cyprinus carpio when fed OTC feed at a higher dose (15 g/kg live weight). In contrast, the oral administration of low levels of OTC (150 mg OTC/kg feed) caused no alteration of the kidney tissues of Piaractus mesopotamicus (Carraschi et al. 2012). The present study also noted inflammation in the renal tubules of OTCdosed O. niloticus upon extended dosing, possibly due to the presence of inflammatory cells within the tubule lumen, epithelium or both. The inflammation of renal tubules (nephritis) and disintegrated glomerulus revealed defective glomerular filtration of blood. The inflammation and loss of structural integrity of the renal tubules in OTC-dosed fish may decrease the availability of ATP and impair the energy supply (Baldissera et al. 2017). A perusal of literature on the histopathological changes in the kidney of fish treated with various antibiotics at varied concentrations through different routes of administration indicated conflicting results. For example, several earlier studies reported no changes in the kidney of fish (Fairgrieve et al. 2005;Straus et al. 2012;Shiogiri et al. 2016), while others demonstrated histopathological alterations in the kidney (Reimschuessel and Ferguson 2006;Gaikowski et al. 2013). Our observations on the progressive increase in the renal changes with the duration of treatment validated earlier studies (Reimschuessel and Ferguson 2006;Gaikowski et al. 2013;Limbu et al. 2020;Yang et al. 2020).
The OTC-dosed O. niloticus of the present study exhibited varying degrees of glycogen-type vacuolisation in the liver. Glycogen vacuolisation is a signal of the degenerative process, which can cause an increase in the size of hepatocytes or acute swelling of the organ (Rejeki et al. 2005). It may also result in depletion of the glycogen reserves in the hepatocytes (Wilhelm Filho et al. 2001) and an imbalance in synthesis and release of metabolic substances (Senarat et al. 2015), thus, leading to stress in fish. The vacuolisation of the hepatocytes was severe in 20-day OTC-dosed O. niloticus than in the 10day group, which suggested severe metabolic damage, possibly related to OTC medication. It implied that vacuoles formation is a defence mechanism against OTC toxicity and may be an adaptive change to protect against liver injury (Wolf et al. 2015). The fish of both trials also had degeneration of hepatocytes and cytoplasmic vacuolisation. Besides, the 20day OTC-dosed group had necrotised liver parenchyma, thus confirming the hepatotoxic effect of OTC. Alike, the gill histopathological sections of 1×-dosed O. niloticus revealed varying degrees of changes on the gills upon extended OTCdosing. Changes observed on 10-day OTC-dosed fish were lamellar fusion, curling of secondary lamellae and desquamation. The 20-day OTC-dosed group had more severe pathological changes, viz., lamellar congestion, desquamation, lamellar epithelial lifting, ruptured secondary lamellae, swelled lamellar tip and thinning of secondary lamellae. The gill tissue damages, as reported here due to extended OTC-dosing, may reduce the oxygen uptake and disturb the osmoregulatory functions of the fish. Similarly, tetracycline-or OTCind uc ed p ath olog i cal cha nge s i n rain bow tr out Oncorhynchus mykiss (Rodrigues et al. 2017) and mosquitofish Gambusia holbrooki (Nunes et al. 2015), possibly associated with circulatory disturbances and oxidative stress, have been demonstrated. In contrast, zebrafish Danio rerio exposed to OTC at 10-50-mg/L levels failed to show changes in the gill tissues (Oliveira et al. 2013).

Conclusion
The effects of OTC on the reduced feed intake and biomass and increased mortalities were observed to be dose-dependent. The oral OTC-dosing caused mild to moderate pathological changes in the gills, liver and kidney. With the cessation of dosing, the fish were able to mount adaptive biological responses to overcome the stress. The levels of OTC residues in edible muscle increased in a dose-and time-response relationship and declined upon withdrawal of medicated feed. The OTC residue levels even on day 20 OTC-dosing at 80 mg/kg biomass/day were lower than the MRL permitted by Codex Alimentarius (200 ng/g). The data on the residue clearance can be useful in establishing the safe withdrawal period of OTC for cultured O. niloticus. From the results of the present study, it can be concluded that oral OTC administration at 80 mg/kg biomass/day for 10 consecutive days at 22-26°C did not affect the survival, feed intake and growth performance of O. niloticus significantly. These observations may be of practical significance and provide effective guidance on the use of OTC in O. niloticus culture.